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Eukaryotic Genomes (4-7) Flashcards

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1
Q

How much human DNA is non-coding?

A

More than 50%
→ 1.5% is exons - coding part
→ as genomes get larger, an increasing proportion of the DNA is non-coding

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2
Q

What makes human genomes so large?

A
  1. Gene duplication → families of genes and pseudogenes with often coordinated regulation
  2. Large introns → often containing retrotransposons
  3. Transposons → ability to change position in genome - LINES, SINES, retroviruses, retrotransposons
  4. Repetitive DNA → simple sequence repeats, segmental duplications
  5. Non-repetitive DNA
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3
Q

What does gene duplication lead to?

A

Protein-coding genes have relative with which they share common ancestry
→ some exist in families and super-families
→ within a genome, families can be dispersed or clustered
→ maintenance of clusters implies functional co-ordination/regulation
→ expands the amount of DNA

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4
Q

What is the globin gene family?

A

New genes formed by duplication during evolution
→ transcriptionally regulated gene cluster
→ duplicates can mutate individually - start getting divergent proteins

One ancestral globin → duplication → divergence → haemoglobin & myoglobin (different functions)

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5
Q

How does haemoglobin gene expression change from foetal to adult?

A

Foetal → express alpha and gamma
Adult → changes to alpha and beta

Demonstrates one of the functions of expanding genome
→ gives the opportunity for developmental regulation - temporal transcription

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6
Q

What are retrotransposons?

A

Type of genetic element found in genomes - capable of copying themselves into RNA then back to DNA which can be inserted at different locations within the genome

LINEs → long interspersed nuclear elements
SINEs → short interspersed nuclear elements

→ regulate gene expression by affecting chromatin structure, gene transcription and pre-mRNA processing
→ make up >40% of human genome

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7
Q

What is the ‘beads on a string’ observation?

A

Chromatin first observed in 1974
→ visualised nucleosomes separated by linker DNA
→ with the removal of H1 by low salt conditions

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8
Q

What is a nucleosome?

A

145-147 base pairs of DNA wrapped tightly ~1 2/3 turns around a globular protein complex
→ histone octamer: 2 copies of H2A, H2B, H3, H4

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9
Q

What are the 5 major types of histone proteins

A

H2A, H2B, H3 and H4
H1 → linker histone

→ rich in +ve basic aa which interact with the -ve phosphate in DNA

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10
Q

What did in vitro reconstruction of the nucleosome reveal?

A

H3 and H4 dimerise then form a tetramer
→ the stable tetramer wraps DNA in a soluble form leaving holes
→ enough space for H2A and H2B to dimerise and join nucleosome
→ N terminal tails protrude from the nucleosome

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11
Q

How is DNA packed?

A

dsDNA
→ beads on a string
→ packed nucleosomes
→ extended nucleosomes
→ extended chromatin
→ compacted chromatin
→ metaphase chromosome

Each DNA molecule is packaged into a chromosomes 10,000-fold shorter than its extended length

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12
Q

How can you transcribe DNA with the high levels of organisation and nucleosomes present?

A

Nucleosomes must be removed or remodelled to allow for transcription and replication

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13
Q

What is DNA acetylation?

A

Reversible modification of histone tail lysine
→ +ve charge of lysine neutralised - proteins involved in transcription can access more easily
→ creates more open form of chromatin

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14
Q

How does the cell choose which histone to be modified by acetylation to open the DNA for transcription?

A

Gene activator proteins bind chromatin remodelling complex
→ leads to remodelled nucleosomes, histone removal or histone replacement

Can also bind histone-modifying enzymes
→ read predominant chemical modification and copy it to other histones - spread of modification down the length of DNA

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15
Q

What is DNA methylation?

A

1/2/3 methyl group added to aa

Specific lysine and arginine residues can be modified by methylation
→ can relax or compact chromatin depending on: which residue is methylated and the degree of methylation (context dependant)

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16
Q

What are some other histone code modifications?

A

Phosphorylation → some serine and threonine resides - promotes transcription
Ubiquitylation → some lysine residues on H2A are recruitment sites for DNA repair, some are activating and on H2B are repressive
Citrullination → some arginine residues on H3 and H4

→ complex histone code determines gene expression - not fully understood

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17
Q

How is the histone code maintained during transcription?

A

RNA Pol II stalls at nucleosome A
→ unwinds half of the DNA from nucleosome A
→ transcribed DNA is looped starts to wind around nucleosome A, while unwinding continues

Same nucleosome is transferred from in-front to behind the polymerase (from downstream to upstream as transcription proceeds)

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18
Q

How is the pre-replicative complex formed?

A

The origins of replication are remarked with an ORC (origin recognition complex)
→ Cdc6 binds DNA at each side of ORC and recruits Cdt1
→ they recruit Mcm helices - opens strands of DNA

In G1 the pre-RC is formed but not active

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19
Q

How is the pre-replicative complex activated in S phase?

A

Cdk2 phosphorylates Cdc6 → falls off and doesn’t recruit Cdt1
→ opens the origin for one round of replication
→ allows recruitment of primes clamp loader and DNA polymerases forming the active replication complex

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20
Q

How is the histone code maintained during replication?

A

The replication machinery displaces H2A-H2B dimers
→ H3-H4 tetramers displaces and reused?
→ not clear but both new strands inherit them

Another set of histones is made to fill in the gaps
→ immediately acetylated
→ NAP-1 and GAF-1 chaperones guide histones into the available spaces
→ produces rebuilt histones - acetylated keeping structure ope

Acetylation is removed then histone-modifying enzyme/reader-writer complex copies epigenetic information
→ maintenance of histone code
→ transfer of epigenetic history

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21
Q

What is epigenetics?

A

Heritable changes in gene expression that are not mediated at the DNA sequence level

Chromatin modifications
→ DNA methylation
→ histone modification

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22
Q

How can SINEs regulated gene expression?

A

Non-epigenetic → act as enhancers or alternative promoters
→ recruit transcription factors and promote expression of nearby genes

Epigenetic → GC-rich so are hot spots for DNA methylation
→ can silence nearly genes stimulating chromatin condensation

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23
Q

Why is transcriptional regulation needed?

A

Allows development of different tissues
Transition from childhood to adult
Deregulation can result in uncontrolled growth (cancers)
Allows reaction to environmental cues

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24
Q

What 3 ways is transcription controlled?

A
  1. Chromatin structure
  2. RNA polymerase (and general TF) binding specificity
  3. Additional binding and activation factors
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25
Q

When is chromatin expressed/silenced?

A

Condensed chromatin → genes with highly packed heterochromatin are usually not expressed - silencing

De-condensed chromatin → expressed genes found in this ‘open’ formation

→ we can open/close chromatin shifting bias between expression and silencing

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26
Q

What is methylation of lysine at position 9 in the histone code associated with?

A

The formation of heterochromatin - gene silencing
→ H3K9me
→ type of heterochromatin depends on whether its tri- or uni- methylated

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27
Q

What is H327me associated with?

A

H3 → histone H3
K27me → lysine position 27 methylated

Associated with inactivation of HOX genes
→ developmental genes arrayed in the same order as the segments int he body - key role in organising body plan of embryo

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28
Q

What are some histone code modifications associated with gene expression?

A

H3S10p H3K14ac → gene expression
H3K4me H3K9ac → gene expression

→ relax DNA, open chromatin structure allowing for gene expression

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29
Q

What is the centromeric histone code modification?

A

H3K4me2 → double methylation of lysine at position 4
→ keeps structure open
→ important for centromere as must be open at all times

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30
Q

What is theorised about the scaffolding holding interphase chromosome in loops?

A

The scaffold is poorly understood but looped DNA assumed to be held by many scaffolding proteins
→ contains lots of topoisomerases for the winding and unwinding of DNA

Modification extends loops for high level of gene expression

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31
Q

Where is chromatin within the nucleus?

A

Heterochromatin → genes tuned off, close to the nuclear membrane associated iwht the lamina where its formed
Active regions → for genes that need to be turned on, the loop is opened up and relaxed
→ then moved to an area where all the requirements for transcription are concentrated

→ there is dynamic movements of genes within the nucleus

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32
Q

What are the two major types of heterochromatin?

A

Facultative heterochromatin → cell-type specific, can switch to euchromatin following developmental cues, characterised by a specific histone code mark
→ H3K27me3 that binds ‘polycomb’ proteins

Constitutive heterochromatin → regions that are consistently silenced in all cell type of an organism - centromeres, telomeres
→ H3K9me3 - carried out by histone methyltransferases (HMT)

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33
Q

How is the human centromere organised?

A

Centric chromatin flanked by peri centric chromatin

Centric chromatin → long highly repetitive chromatin structures

Double methylation of lysine 4 (H3K4me2) → gives centromere open structure allows for kinetochore attachment
→ important for function

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34
Q

What are telomeres?

A

Repetitive DNA sequence found at the end of each of our chromosomes
→ vary in length and repeated DNA sequence
→ vertebrate sequences at over several kb
→ yea several hundred bp

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35
Q

What is the end replication problem?

A

DNA is synthesised 5’ to 3’
→ leading strand - continuous synthesis
→ lagging strand - Okazaki fragments

Gaps usually repaired by DNA ligase
→ but end gap on lagging strand can’t be repaired because DNA synthesis required 3’-OH - there is no primer
→ means one of the strands made in shorter than the template
→ so telomeres get shorter over time and fray

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36
Q

What is the Hayflick limit?

A

The number of times a normal somatic, differentiated human cell population will divide before cell divisions stops
→ between 40 and 60 divisions
→ after the cell becomes senescent
→ in-built ageing mechanism

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37
Q

What is the compensatory mechanism for telomere shortening?

A

Telomerase binds the sing-stranded G-overhang
→ extends the 3’ end of the parental strand using its own RNA subunit as a template (RNA directed DNA synthesis)
→ this provides more room for DNA primate to lay down a primer - which can be extended

(extends our telomeres, gives partial not full compensation for reduction in length of our telomeres)

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38
Q

Telomeres end up with a 3’ unpaired end, why do they not fuse?

A

Exposed OH groups are reactive so the 3’ end must hide somewhere
→ the 3’ end hides from DNA repair mechanisms in sheltering complexes - to prevent fusion of telomeres

Shelterin complex
→ TRF 1 / 2 (telomeric repeat-binding factor 1 / 2)
→ RAP1 (repressor/activator protein)
and others

→ stimulates t-loop formation that displaces the d-loop - results in base paining at the 3’ end

39
Q

What is Werner syndrome?

A

Accelerated ageing caused by mutation in in the WRN gene
→ WRN helices protein important for DNA repair and telomeric DNA replication
→ telomeres that are usually normally replicated by lagging strand synthesis are not replicated efficiently in Werner cells

(inheritance: autosomal recessive)

40
Q

What do human Pol II promoters typically look like?

A

Contain multiple cis-activing elements that bind proteins

BRE^u → upstream B recognition element - binds TFIIB
TATA box → binds TATA- binding protein (TBP)
BRE^d → downstream B recognition element - binds TFIIB
Inr → initiator element binds TFIID

Individual elements not always present → Pol II promoters are very variable

41
Q

What is the basal transcription apparatus?

A

TFIID complex binds TATA box via TBP, aided by TFIIA

TBP recruits TFIIB which recognises BRE^u and BRE^d and accurately positions RNA Pol II at the start site of transcription

TFIIE, RNA Pol II?TFIIF are recruited
TFIIF → stabilised RNA Pol II interactions with TFIIE and TFIIH

TFIIH is recruited by TFIIE

→ won’t move until further signals

42
Q

How is elongation initiated?

A

TFIIH complex → helicase opens DNA double helix
→ kinase phosphorylates the C’ - terminal domain of the RNA Pol II L’ subunit

RNA polymerases don’t require a primer - elongation starts

RNA Pol II now disengages from the cluster of general transcription factors - undergoes conformational change that tightens its interaction with DNA

→ phosphorylation of the CTD marks the transitions from initiation to elongation

43
Q

How can polymerases binding to the TATA box regulate transcription?

A

Different TATA boxes are recognised with different affinities
→ some are more efficient at stimulating transcription can others

44
Q

What is the G-less cassette transcription assay?

A

A promoter is cloned upstream of a G-less cassette (made from A, C, T bases only)
→ purified TES, RNA Pol II, ATP, CTP and [alpha^32P]-UTP added

Result → radioactive RNA transcript of a defined size
→ can be electrophoreses through polyacrylamide gels and quantified following autoradiography

No GTP is supplied the RNA is truncated at the point which a ‘G’ should be inserted

45
Q

What are some additional binding factors that can control transcription?

A

Activator/repressor proteins
Mediator proteins
Chromatin-modifying proteins

46
Q

What are homeodomains?

A

Type of DNA-binding domain that can help regulate gene expression
→ Helix 3 binds the major groove of DNA making specific interactions between aa and nucleotides

→ shape and DNA binding is conserved

47
Q

What are zinc finger motifs?

A

A small structural motif with key Cys and His residues that coordinate a zinc ion stabilising the fold
→ 2 beta strands - 2 Sys residues which binds one zinc molecules
→ 2 alpha strands - 2 His residues that bind same zinc molecule
→ interacts with major groove of DNA

Key charged molecules Arg and His make specific contact with G residues - often cluster in DNA
→ GC rich areas prime binding sites for zinc finger motifs
→ demonstrates sequence specificity

48
Q

What are leucine zippers?

A

Type of DNA-binding domain
→ cross-shaped binding to specific sequences

49
Q

What is multimerisation and combinatorial control?

A

Combinatorial regulation is a powerful mechanism that enables tight control of gene expression
→ via integration of multiple signalling pathways that induce different transcription factors required for enhanceosome assembly
→ all 3 pathways activated - high levels of expression
→ depending on stresses cell is responding to

50
Q

What is an enhanceosome?

A

Complex formed by the binding of multiple transcription factors to specific DNA sequences - enhancers
→ cooperative binding of different proteins to forma fully active enhanceosome
→ enhancer recruits high-mobility group (HMG) proteins which regulate chromatin structure - ensure the target gene can be accessed by the transcription factors
→ make the promote accessible
→ tight regulation of gene expression

51
Q

What is the Philadelphia chromosome?

A

Gene abnormality found in some individuals with certain types of leukaemia
→ results from a gene translocation creating a fusion gene called BCR-ABL1 has abnormal kinase activity so is permanently switched on
→ leads to unregulated cell proliferation

52
Q

What is Burkitt’s lymphoma?

A

A type of non-Hodgkin lymphoma caused by inappropriate regulation of an oncogene by a very strong enhancer

53
Q

What are some eukaryotic Pol II post-transcriptional events?

A

RNA Pol II transcription
→ possible attenuation (RNA transcript aborts)
→ 5’ capping
→ splicing
→ 3’ capping
if these fail leads to non-functional RNA sequences - degraded in the nucleus
→ possible RNA editing
→ nuclear export

not all events occur for each transcript
→ they require factors that bind the phosphorylated C-terminal domain (CTD) of RNA Pol II

54
Q

What marks the transition from transcriptional initiation to elongation?

A

Phosphorylation of the seryls 2 and 5 on the CTD
→ acts as recruitment sites for enzymes that modify the transcript
→ the CTD of Pol II comprises multiple repeat regions of a 7aa sequence YSPTSPS

55
Q

How is the 5’ cap added to RNA transcripts?

A

RNA triphosphatase removes one phosphate from the nascent RNA transcript
→ leaves site for guanylyl transferase to cleave a diphosphate off a GTP and attach it to the remaining P pair
→ this is then modified by methyltransferases
→ forms an unusual 5’to5’ triphosphate bridge

Result: cap put on backwards so 5’ end can mascaraed as th 3’ end → no longer vulnerable to degradation by 5’ exonuclease

56
Q

What are the capping enzymes?

A

RNA triphosphatase
Guanylyl transferase
Methyltransferases
→ recruited by the phosphorylated CTD of Pol II
→ 5’ capping is co-ordinated with transcription

57
Q

What is the purpose of a 5’ cap?

A

Characterises Pol II transcripts from other RNA molecules → no other transcripts are modified like this
Stabilised the RNA → there is no 5’ phosphate so its resistant to 5’ exonuclease
Aids in further processing and export to the cytosol
Required for efficient translation of mRNAs

58
Q

How are capped mRNAs regulated?

A

Decapping
→ 5’ caps protect mRNAs from exonuclease activity
→ regulation depends on (in part) decaying pathways

Multi subunit cytosolic ATP-dependant decapping enzyme complex
→ removes the 5’ cap
→ restores the 5’ phosphate
→ venerable to 5’ exonuclease
→ mRNA can no longer interact with ribosome - stops protein synthesis

Regulated decapping controls the half-life of a mRNA

59
Q

How was it determined that RNA itself is catalytic?

A

Firstly an intron was identified, then it was found to be excised from the primary transcript
→ wanted to see what enzyme did this

Cech and his team
→ added phenol - denature protein
→ and boiled
intron still accumulated

→ allowed them to come to the conclusion that RNA could catalyse functions previously thought to be enzyme functions - intron described as a ribozyme

60
Q

How was the splicing mechanism discovered?

A

Cech and his team used ‘Tetrahymena’ rRNA transcripts

61
Q

How do human mRNA transcripts undergo splicing?

A

Use an inbuilt co-factor (branch site adenosine)

  1. The RNA folds by base pairing → creates a complex structure placing the branch site hydroxyl in a position where it can attack the phosphate at the 5’ splice junction
  2. This adenosine now has 3 phosphodiester bonds → one is a 2’, 5’ phosphodiester bonds - circularises the intro into lariat structure
    → the 3’ OH of the upstream exon (nucleophilic) attacks the phosphate at the 3’ splice site
  3. Exons are fused and the intron is removed as a lariat
62
Q

What protein complex controls splicing?

A

By the spliceosome: an RNA and protein complex
→ 5 snRNPs (small nuclear ribonucleoproteins) U1, U2, U4, U5, U6 - small nuclear RNA complex with at least 7 protein subunits

Splicing must be precise so spliceosome recognises specific sequences at:
→ the 5’ splice site
→ the branch site
→ the poly pyrimidine tract (poly Y)

Base-pairing specificity → allows assembly of spliceosome
Other proteins → stabilise, provide other functions
→ both allows precise splicing as splice sites and branch site are bought close together

63
Q

How does the spliceosome work?

A
  1. U1 snRNP binds the 5’ splice site → base pairing provides specificity
  2. Branch binding protein (BBP) and the protein U2 auxiliary factor (U2AF) bind the are that encodes branch site adenosine and associated pyrimidine tract
  3. U2 is recruited → displaces BBP
  4. A pre-formed trimer of U4, U5 and U6 associates
  5. A molecular rearrangement (loss of U1 and U4) activates the complex → 5’ splice site is exposed and can be attacked by the branch site adenosine
  6. The 2’-OH attacks the 5’ splice site phosphate
64
Q

Which sequences are recognised by the spliceosome?

A

U1 only recognises 9 bases → only 3 of these are highly conserved
→ lots of different strengths of sequences identifying the 5’ splice site

Splicing signals are consensus sequences → individual motifs have different affinities for spliceosome components
→ some are more efficient at stimulating splicing than others

65
Q

How are the U1, U2, U4 AND U5, snRNPs made?

A
  1. Transcription by Pol II, capped and shipped to cytosol
  2. In cytosol 7 Sm proteins are loaded in a ring onto the snRNA by SMN protein
    → cap hypermethylated - signal to return to nucleus
  3. Transported to nucleus and maturation into e.g. U1 snRNP by association with other proteins

(U6 lacks Sm proteins and remains nuclear)

SMN → Survival Motor Neuron

66
Q

What is the ‘exon definition’ hypothesis?

A

Exons are defined by the binding of proteins during transcription → allows U1 to find the 5’ splice site - how it recognises just a few bases
→ if you have a defines end of the first exon by the binding of SR protein as the U1 is scanning its only got to find a suitable site next to a SR protein
→ provides the added specificity that allows U1 to bind

Long exons have hnRNP to inhibit splicing

67
Q

What is the role of the poly Y tract in alternative splicing?

A

A strong consensus poly Y tract → 3’ end of intron strongly defined - strong defining of where U2 binds
→ normal splicing

A weak consensus poly Y tract → sometimes U2AF binds, BBP binds, U2 recruited, 3’ end defined - normal splicing
→ sometimes U2AF disengages and U2 not bound, 3’ end not defined - alternative splicing/exon skipping g

→ alternative splicing is regulated by the strength of the poly Y tract

68
Q

How can alternative splicing be controlled in a cell-type specific manner?

A

U2AF is a family of proteins whose members have different abilities to find poly Y tracts
→ generates protein control which can be controlled developmentally

Different cells express different versions → example of a need for weak ineractions

e.g. mRNA in thyroid cells produces calcitonin but in neuronal cells produces calcitonin gene related peptide - one gene: more than one protein

69
Q

What is haemophilia?

A

A blood clotting disorder caused by mis-splicing
→ mutation causes A to G transition in intron 3 of the F9 gene
→ creates a false 3’- splice site
→ leads to mis-splicing producing a defective protein

70
Q

What is spinal muscular atrophy (SMA)?

A

During the biogenesis of snRNPs Sm proteins are loaded onto the snRNA by SMN (survival motor neuron) protein
→ there are 2 versions of SMN (1 does 90% and 2 does 10% of loading)
→ most of the SMN2 protein made is non-functional because the mRNA was mis-spliced
→ SMN deficiency caused by SMN1 mutation leads to widespread splicing deficiency - especially in motor neurons

71
Q

What happens at the 3’ end of a pre-mRNA?

A

Addition of a poly (A) tail

72
Q

How is the poly(A) tail added to pre-mRNA?

A
  1. Recruitment of the cleavage factors → poly(A) site bound by CPSF, cleave site CF, G/U rich region CStF
  2. Interactions between CStF and CPSF provide a recruitment site for the polymerase (PAP)
  3. Conformational change exposes cleave site → RNA cleaved by CF
    → provides a 3’-OH for PAP
  4. PAP adds adenine residues → slow adenylation until ~12 added
  5. Poly(A) binding protein binds → switch from slow to fast adenylation for ~200, then PAP disengages
73
Q

What are the functions of polyadenylation?

A

Nuclear export
Translation
Stability of mRNA

74
Q

What is RNA editing?

A

Many RNAs need to have their bases changed
→ occurs in eukaryotic tRNA, rRNA, mRNA and miRNA, archaea and bacteria (universal)
→ in trypanosomes there is extensive RNA editing of some kinetoplast genes
→ in vertebrates RNA editing occurs in the nucleus

75
Q

In what species does the most dramatic gene editing occur?

A

Trypansoma brucei
→ the mitochondrial DNA (kinetoplast DNA) contains a mixture of maxicircles and minicircles interlocked
→ the maxicircle kDNA encodes two rRNAs, one ribosomal protein and seventeen protein-coding sequences - 12 of the ORFs require RNA editing in order to be converted into translatable mRNAs
→ some maxicircle mRNAs are extensively edited - many Us added
→ some maxicircle genes are encrypted - transcripts must be decrypted before being read

76
Q

How do trypanosomes modify their RNA transcripts?

A
  1. GuideRNA 1 encoded on a minicircle binds the target RNA by pairing at its 5’ anchor and 3’ tail sequences
  2. Specialised proteins recognise the addition/deletion sites and add/remove Us → copying the information from the gRNA - resulting in an extended mRNA
  3. The editing provides an anchor site for gRNA 2 - encoded on a different minicircle → same process repeats

→ every loop on the gRNA has an A - determines where Us will be inserted

77
Q

Why do mitochondria of trypanosomes and plants utilise extensive RNA editing?

A

Its speculated that different guideRNAs are expressed in different environments
→ is RNA editing a primitive way to change the expression of genes?

78
Q

What is mammalian nuclear APOBEC-mediated mRNA editing?

A

Cytosine deaminated to generated uracil
→ change of amine to oxygen

→ involves an editosome

79
Q

What does an editosome consist of?

A

APOBEC (ApoB mRNA editinf enzyme catalytic subunit) - 2 domains - catalytic / pseudo catalytic
→ pseudo catalytic - turns itself off until its associated at the site of deamination along with an auxiliary complex (ACF - APOBEC complementation factor)
→ the combination (editosome) recognises RNA sequences around the editing site then specifically edits

80
Q

What is the function of the APOBEC editosome?

A

RNA editing of Apo-B gene - C to U editing on exon 26

(ApoB required for uptake and transport of cholesterol)

Liver → no editing - ApoB-100, lipid-associated, binds LDL receptors

Intestine → strong expression of APOBEC thus there is C to U editing - creates stop codon creating shorter version of ApoB
→ ApoB-48 - lipid-associated, doesn’t bind LDL receptors

→ one gene, two proteins/functions - separates by cell-specific expression

81
Q

What is mammalian nuclear ADAR-mediated mRNA editing?

A

Adenosine deaminated to inosine by ADAR (adenosine deaminase acting on RNA)
→ amine to oxygen
→ iodine is a mimic guanosine

ADAR requires dsRNA → binds a fold back i.e. stem loop structure

Exon → I is translated as G - can change protein sequence
Intron → I mimics G - can create new splice sites
→ ADAR driven editing can alter splice sites - regulate alternative splicing and protein function

82
Q

Why would there be inverted repeats in our mRNA?

A

Imagine 2 Alu elements inserted into opposite strands of DNA → when mRNA is made complementary base pairing causes foldbacks (allows ADAR editing)

Alu elements → a class of primate-specific retrotransposons ~300 bases long - sub-group of SINEs

There are more than one million Alu elements dispersed in out genomes → provides plenty of opportunity to derive dsRNA

83
Q

What is the value of ADAR-mediated RNA editing?

A

May have evolved as a defence system to inactivate retroviruses/retrotransposons → protects our genome

Enhances genome plasticity → generate new protein functions

ADAR can recognise DNA:RNA hybrids → role in DNA repair

Neurobiology link to epilepsy

84
Q

How is mRNA edited for nuclear transport?

A

Export factors are loaded on to a mRNA during transcription by the C-terminal tail of one of the large subunits of RNA Pol II
→ nuclear export is fully integrated into mRNA maturation

At a late stage in splicing a nuclear export receptor complex is transferred from the CTD of Pol II
→ provides a nuclear export signal (NES) allowing message to be exported to the cytoplasm

mRNP - complex compacted particle of mRNA and proteins → mature enough to be exported

85
Q

How does nuclear export of mRNA occur?

A
  1. The nuclear export signal (NER) binds nuclear cage around a nuclear pore
    → mRNP is threaded through 5’ end first, SR proteins removed and recycled
  2. The cap binding protein (CBC) is removed → replaced by eukaryotic initiation factor 4E (elF4E) and circularised with elF4G (NER returned to nucleus)
  3. The mRNP is now ready for translation
86
Q

Why is miRNA of high importance?

A

microRNA action in cytosol still being fully understood

In 2020 → ~500 genes controlled by miRNAs
In 2024 → 1/3 eukaryotic genes regulated by miRNA

Role of miRNAs in regulating gene expression might be as important as that of transcription factors

87
Q

What are miRNAs?

A

MicroRNA → small non-coding regulatory RNAs processed from dsRNA precursors
→ interfere with expression of other RNAs
→ typically stem-loop ds structure
→ 2-base overhang at the 3’ end
→ GC-rich - stable structure

88
Q

What is involved in the biogenesis of miRNA?

A

Canonical pathway → miRNA precursors can be found inter genetically (sometimes in clusters)
→ primary miRNAs formed
→ Drosha in the nucleus makes specific cuts at cleavage sites forming pre-miRNA

Non-canonical pathway → encoded in an intron (a mitron)
→ mitron lariat formed - debranching and base-pairing forms pre-miRNA

89
Q

How are pre-miRNAs exported from the nucleus?

A

Processed pre-miRNAs don’t have a 5’ cap nor a poly-A tail → required exporting 5/Ran GTP

Exportin 5 binds the pre-miRNA (required the 2bp overhang)
→ associates with Ran GTP - allows export through the nucleus
→ complex falls apart in the cytosol
→ miRNA stays in cytosol - other components recycled

90
Q

How is RISC (RNA-induced silencing complex) formed?

A

Cytosolic pre-miRNA binds Dicer ( a cytosolic RNAse III family member) → forms a mature duplex: guide strand and passenger strand

Cytosolic chaperones (Hsp90/70) bind argonaute → change conformation to open - can accept miRNA

Mature duplex loaded onto AGO, passenger strand degraded → left with RISC - a regulatory complex that can regulate expression of other genes

91
Q

What is the role of RISC?

A

If the guide RNA of RISC has strong homology to the target gene → will bind strongly to the mRNA
→ argonaute slices it in 2 - leaves 2 sites for nucleases to attack
→ also recruits a deadenylase
→ degradation of mRNA

If the guide RNA has weak homology to target mRNA
→ translation inhibition

Shuts down gene expression of target gene

92
Q

What are some examples of miRNA action?

A

miR-184 inhibits Numbl
→ controls neural stem cell differentiation - miR-184 antagonises and stops differentiation

miR7 / miR-206 inhibit Pax7
→ muscle cells

93
Q

What can happen when miRNAs go wrong?

A

Dysregulation of protein expression

Severe developmental defects e.g. in limb formation, heart defects

Cancer

Neurological defects, behavioural effects

Infertility

Dicer is inactivated in fertilised eggs, blocking the generation of all miRNAs at this developmental stage

Molecular Cell Biology (6 decks)

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